Vitamin k compounds and process of obtaining same



@atenteol Apr. 16, 1945 awa ts VITAMIN K COMPOS PRUUESS F OBTG Sm Edward A. Doisy and Donald W. MacComuodale,

Webster Groves, and Sidney A.

Stephen B. Binkley, and Ralph W. McKee, St. Louis, Mo., assigncrs to President and Board of Trustees of St. Louis University, St. Louis,

Mo. I No Drawing. Application-June '1, i939,

' 1 Serial No. 271,774!

13 Claims. (Cl. 266%88) The invention relates to a method of obtaining compounds having antihemorrhagic or vitamin K activity, whereby intermediate compounds of greater stability than the vitamins from which they are derived are produced.

It is known that vitamin K products and concentrates areobtainable from various animal and vegetable materials, such as alfalfa, spinach, soybean oil, liver, putrified fish meal, etc. On the other hand, it is also known that the antihemorrhagic vitamins are labile substances which are subject to inactivation in, greater or lesser amounts during the usual processes of extraction, adsorption, distillation, etc., used for isolating them.

. The present invention overcomes,- to a great extent, the objections mentioned in the processes heretofore used, by reacting the vitamin K in its relatively crude form with an esterifying or etherifying agent, either during or after reduction of quinone groups to hydroquinone groups, to obtain an ester or an ether of the reduced'form of the vitamin which, because it is more stable than the vitamlmis then subjected to the known methods of concentration and purification with practically no loss of activity. After the reduced and esterified, or etherified, derivative has been sumciently concentrated, it can be converted back into a purified vitamin product by hydrolysis and oxidation. Instead of converting the derivative back into the vitamin from which it was obtained, the derivative can, in many instances, be directly used for therapeutic purposes, since it has been found that the derivatives often have high antihemorrhagic potency, in some cases having activity of the same order as the original vitamin itself.

The invention is not limited to the use of crude or impure vitamin K products as starting materials, since it is possible to start. with a pure vitamin K, as obtained for example by isolation from a natural source or by synthesis, convert this by reduction and esterification or etherification into the ester or ether, and use the latter for its vitamin K efiect. An advantage in such cases is that the ether or ester obtained has greater stability than the natural vitamin from which it was derived.

Vitamin K activity is exhibited by a number of difierent substances. However, apparently all of them have a quinone-Iike chemical structure, and are capable of reduction to hydroquinonelike compounds. The present invention can therefore be more fully understood by considering the esterification or etherification step of the present the vitamin by groups capable of hydrolysis to ing examples:

groups in'the reduced or hydroquinone form of The starting material tor this example is vita min K1, a pure antihemorrhagic vitamin described in our publication in the Journal of the American Chemical Society, 61, 1295, and ob-" tained by petroleum ether extraction of dried invention as a replacement of phenolic hydroxyl alfalfa meal and subsequent purification by adsorption on a zeolite. Such a product is hereinafter designated as vitamin K1. It is an oil at ordinary. temperatures, having a p'olyene struc ture which absorbs 4 moles ot-hydrogen' upon catalytic reduction, exhibits ultraviolet absorption spectrum maxima (hexane solution) at243, 248, 261, 270, and 323 my. and has an antihemorrhas'ic potency of approximately 1 chick unit per 0.5 microgram.

milligrams of this vitamin K1 are dissolved along with 101) mg. or iused sodium acetate in lll cc. of acetic anhydride. The-solution is then re fluxed with 1 gram or zinc dust ior 30 minutes.

Anadditional l g; of zinc dust is added, a few milligrams! at a time, d theperlod of heat ,ing; The mixture is filtered ml abut. "the acetic anhydride filtrate is decomposed by the addition of 20 to 30 cc. oi waten- After deccmposition has taken place, the nit: v e is extracted with ether, the ether extract is washed with water and evaporated to dryness. l'he residue is then taken up in and recrystallized from absolute methyl alcohol at C. The product can also be crystallized from low boiling petroleum ether (30-60" C.). It is very soluble in acetone, ethyl alcohol and benzene and can be recrystallized in the form of fine snow white needles from methyl alcohol or petroleum ether. It has a melting point of 59C. and is pure diacetyl dihydro vita K1. Analysis of the compound gives the following figures:

Carbon" -per cent" 78.21 and. 78.01 Hydrogen do. 10.07 and 10.03 Oxygen per cent" 11.72 and 11.96 (by difierence) Molecular weight 531 (East) Microhydrogenation of a sample of the diacetyl dihydro vitamin K1 causes it to take up 3.04 moles of hydrogen (H2) The vitamin K1 itself took up 4.08 moles of hydrogen.

illustrated by the follow- Bio assay by the chick method shows that the diacetyl dihydro vitamin K1 has an activity of about 500 units per mg.

Absorption spectrum examination of thediacetate of this example shows a general absorption in the region from 220 ma to beyond 300 m with intense absorption at 230 ma where the extinction coefficient is The diacetate of this example is not readily hydrolyzed by alkali 'or acids in an aqueous or alcoholic medium. In alcoholic solution its activity is not destroyed by 100 hours exposure to the light from a 100 watt bulb at a distance of 4 feet.

On the other hand, there is extensive destruction of the vitamin activity when vitamin K1 itself is exposed under the same conditions and for the same length of time.

The diacetyl dihydro vitamin K1 'of this example is convertedto vitamin K1 by treating an anhydrous ether solution of'the diacetyl dihydro Carbon per cent 82.34

Hydrogen do 10.13 1 Oxygen per cent 7.53 (by difference) It has an antihemorrhagic activity in the chick test of 1,000 units per milligram.

That the. vitamin K1 produced by hydrolyzing the dihydro vitamin K1 diacetate obtained in this example is identical with the pure vitamin K1 started with is further proved by the fact that reductive acetylation can be repeated on it to give a diacetate of a dihydro vitamin having a melting point and giving results in a bio-assay identical with the original diacetate of dihydro vitamin K1. The diacetate from the second reductive acetylation also does not give any lowering of the melting point in a mixed melting point test with the original diacetate of dihydro vitamin K1.

Example 2 The starting material for this example is a petroleum ether extract of dried alfalfa leaf containing about 100 chick units of vitamin K1 per milligram of solids. 250 mgs'. of this concentrate are dissolved with 250 mg. of fused sodium acetate in cc. of acetic anhydride; The solution is then refluxed with 3 gms. of zinc dust for minutes. An additional 2 gms. of zinc dust is added, a few milligrams at a time, during the period of heating. The mixture is filtered while hot, the acetic anhydride filtrate is decomposed by the addition ,of 80-90 cc. of water. After the anhydride has posed by adding cc. of water.

been decomposed, the mixture is extracted with ether, the ether extract washed and evaporated I 4 calcium carbonate, etc., which are known to cause wt-ensive destruction of vitamin activity when used with vitamin K1 itself. Using a column of 'Permutit 35 mm. in diameter by 40 cm. long, the

cc. of petroleum ether solution of the crude diacetate of dihydro vitamin K1 is poured onto the top of the column, followed successively by about 2 liters of petroleum ether, 1 liters of petroleum ether containing 10% of benzene, 1 liter of petroleum ether containing 20% benzene, 1 liter of petroleum ether containing 50% benzene, 2 liters benzene and 1 liter alcohol, collecting 500 cc. fractions at the bottom of the column. The purified diacetate of dihydro vitamin K1 is found chiefly in the benzene fractions while most of the inactive material is obtained in earlier fractions. The solvent is removed from the active fractions, and the residue is taken up in and crystallized at low temperature from absolute methyl alcohol. After recrystallizing a few times it melts at 59 C. and is identical with the diacetate of Example 1. By combining the least active fractions and passing them through the column again it is possible to obtain practically quantitative recovery, of all the vitamin activity in the form of substantially pure diacetate of dihydro vitamin Kl.

Example 3 Material for this example is designated as vitamin K11 and. is a crystalline product melting at 52 C. described in our publication in the Journal of the American Chemical Society, 61, 1295, and obtained by extracting putrified fish meal with an organic solvent and treating the extract with a zeolite adsorbent.

200mg. of vitamin K2 is dissolved along with 200 mg. of fused sodium acetate in 22 cc. of acetic anhydride. The solution is refluxed with 2 gm. of zinc dust for 30 minutes, adding an additional gram of zinc dust, a few milligrams at a time, during the period of heating. The hot mixture is filtered and the acetic anhydride filtrate decom- After the anhydride has reacted with the water, the mixture is extracted with ether, the ether extract washed with water and evaporatedto dryness. The residue is taken up in and recrystallized from cold absolute methanol and the diacetate of dihydro vitamin K2 separates as a white crystalline prod-.

uct melting at 57-58 C.

The diacetate gives the following analysis: 7

Carbon per cent 80.89 and 81.03 Hydrogen do 9.94 and 9.979

Oxygen percent 9.17 and 8.99 (by difference) Molecular weight 628 li...= 1280 at 232 1",. Example 4 This example is carried out as described under Example 2, but using as starting material a crude petroleum ether extract of putrified fish meal containing chick units of vitamin K2 active material per milligram of solids. After the solvent is evaporated off of this extract, the crude vitamin K2 residue is converted to the crude dihydro diacetate by reductive acetylation and then purified by an adsorbent as described under Example 2.

The diacetate products of Example 2 and of Examples 3 and 4 can be hydrolyzed and oxidized to get back pure vitamin K1 and vitamin & respectively, in the same manner as described under Example 1. Instead of using sodium acetate and acetic anhydride, other alkali metal carboxylates and carboxylic acids can be used. For example, by using a propionic acid, benzoic acid, chloroacetic acid, or succinic acid anhydride, alongwith the corresponding alkali metal salt, one can obtain the dihydr vitamin propionates, chloroacetates, succinates, etc.

The examples show that vitamin K which has been converted to its phenolic form and esterified or etherified is more stable than in its original quinone-like form. It can thereafter be treated,

in accordance with the present invention, by any of the known methods .of concentration and purification, such as adsorption, fractional distillation, fractional crystallization, etc., with practically no loss of vitamin activity.

Although the examples show simultaneous reduction and esteriiication of the vitamin, the invention also embodies the variation wherein the quinone-like vitamin is first reduced and then, esterifled or etherifled. The two variations of the process canbe represented diagrammatically as follows:

I .Quinone form olvitamin K Reduetive Diester oithe .v 8 nine 6 i I Esterification t i itemi ii K Mild Oxidation H Concenltrete Activity. Hy uinone loan H drol fitnmin K I Purified Diester Redue Esterll Quinone form H i Kym Di ter ---a one arm -v as or of vitamin K y Etherliy Diether of Hydroquinono 4 iorm MildIOxldation Conceal irate Activity H dro H droflninone form Purified Diester o! tamin K or Dioiher Reduction to the hydroquinone formv can be accomplished by any,of the known methods for converting a quinone to a dihydric phenol. For

example, catalytic reduction or hydrogenation may be used. Also, reduction with sulfur dioxide, sodium hydrosulflte, titanous chloride, stannous chloride, 'a'nd the like can be used.

What we claim as our invention is: 1. Process for obtaining an antihemorrhagic compound comprising treating a compound of the class ofvitamins Kr and Relieving-a,

quinone-like chemical structure to reduce said compound to its hydroquinone form and to replace the phenolic hydroxyl group of the latter by a group capable of hydrolysis to give a hydroxyl group.

2. Process according to claim 1 in which the vitamin compound treated is vitamin K1.

3. Process according to claim 1 in which the vitamin compound treated is vitamin Kg. 10 4. Process according to claim 1 in which the reduction and replacement of the hydroxyl group are simultaneous.

5. Process for obtaining an antihemorrhagic compound comprising reacting a compound of the class consisting of vitamins K1 and K2 having a quinone-likechemical structure with a reducing and etheriiying agents to obtain a compound of the class consisting of esterified and etherifled dihydro derivatives of vitamins K1 and K2.

6. An ester of a compound of the class consisting of dihydro vitamin K1 and dihydro vitamin K2 in which the phenolic hydroxyl groups have been replaced by organic carboxylic acid groups :capable of hydrolysis to give hydroxyl groups.

' '7. An ester of dihydro vitamin K1 in which the phenolic hydroxyl groups have been replaced by organic carboxylic acid groups capable of hydrolysis to give hydroxyl groups.

8. An ester ofdihydro vitamin K2 which Y the phenolichydroxyl groups have been replaced by organic carboxylic acid groups capable of hydrolysis to give hydroxyl groups.

bstituted organic lower carboxylic .9. An acid ester of dihydro vitamin K1.

10. An unsubstituted organic lower carboxylic acid ester of dihydro vitamin K2. 11. Diacetyl dihydro vitamin K1 having a melting point of approximately 59 C.

l2. Diacetyl dihydro vitamin K: having a melting point of approximately 57-58 C.

1 13. Process for obtaining an antihemorrhagic I compound comprising treating a compound of the class consisting of vitamins K1 and m with zinc dust and acetic anhydride to obtain the dlacetate of a dihydronaphthoquinone corresponding to the original vitamin K compound.

- EDWARD A. DOISY. 1

DONALD W. MecCORQUODALE. SIDNEY A. THAYER'.

STEPHEN B. BINKLEY. RALPH W. McKEE. 

